Molecular characterization and liquid chromatography-mass spectrometric multiple reaction monitoring-based detection in case of suspected phalloides syndrome poisoning

• Main Authors: Sathaporn Ramchiun, Sujitra Sikaphan, Siriwan Leudang, Dutsadee Polputpisatkul, Nattaphong Nantachaiphong, Thitima Khaentaw, Nattakarn Nooron, Chutimon Uttawichai, Sittiporn Parnmen
• Format: Article
• Language: English
• Published: Chaing Mai University 2018-11-01
• Series: Journal of Associated Medical Sciences
• Subjects: Amanita; internal transcribed spacer region; LC-MS/MS; phalloides syndrome
• Online Access: https://www.tci-thaijo.org/index.php/bulletinAMS/article/view/142631
• ISSN: 2539-6056; 2539-6056

Backgrounds: The most common lethal mushrooms are invariably attributed to amanitas, which contain several types of lethal peptide toxins. During 2015 to 2018, the suspect in phalloides syndrome case reported to the Thai National Institute of Health included 33 patients with 3 deaths. This syndrome is characterized by a long latent period and having two phases of gastrointestinal irritation followed by progressive liver dysfunction. Objectives: The aims of this study were to identify mushroom samples from four clinically reported cases based on nuclear internal transcribed spacer (ITS) sequence data and diagnose lethal peptide toxins using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) with multiple reaction monitoring (MRM). Materials and methods: Nucleotide similarity was identified using BLAST search of the NCBI database. Phylogenetic analysis of nuclear internal transcribed spacer (ITS) region was conducted by maximum likelihood method. Mushroom peptide toxins were analyzed using LC-MS/MS with MRM. Results: Based on BLAST search yielded 98% to 100% of mushroom samples from four clinically reported cases to Amanita brunneitoxicaria. Phylogenetic analysis showed all mushroom samples placed closely related to A. brunneitoxicaria with a strong bootstrap value (BS=100%). The presence of three lethal peptide toxins in clinical mushroom samples was confirmed by MS/MS spectra acquired from a reference standard, including α-amanitin (m/z 919.0, RT=2.157 min), β-amanitin (m/z 920.1, RT=2.167 min) and phalloidin (m/z 789.3, RT=2.189 min). The product ions of m/z 259.3, 259.0 and 330.3 were the most abundant and stable ions for the α-amanitin, β-amanitin and phalloidin analyses, respectively. Conclusion: This study revealed that the toxic mushrooms ingested by patients were confirmed to be a species of amanitas closely related to Amanita brunneitoxicaria. Furthermore, rapid detection using a high-throughput LC-MS/MS with MRM represents an effective method in identifying lethal peptide toxins from poisoning caused by mushrooms.